Prevention and treatment of sepsis

ABSTRACT

Compositions and methods are described for treatment of sepsis in animals, including humans. Unique and specific combinations of polyclonal antibodies directed to cytokines are shown to have a beneficial effect in animal models predictive of human therapy.

FIELD OF THE INVENTION

The present invention relates to therapeutics for the prevention andtreatment of blood-borne and toxin mediated diseases, and in particularthe prevention and treatment of sepsis in humans as well as otheranimals.

BACKGROUND OF THE INVENTION

Sepsis is a major cause of morbidity and mortality in humans and otheranimals. It is estimated that 400,000-500,000 episodes of sepsisresulted in 100,000-175,000 human deaths in the U.S. alone in 1991.Sepsis has become the leading cause of death in intensive care unitsamong patients with non-traumatic illnesses. [G. W. Machiedo et al.;Surg. Gyn. & Obstet. 152:757-759 (1981).] It is also the leading causeof death in young livestock, affecting 7.5-29% of neonatal calves [D. D.Morris et al., Am. J. Vet. Res. 47:2554-2565 (1986)], and is a commonmedical problem in neonatal foals. [A. M. Hoffman et al., J. Vet. Int.Med. 6:89-95 (1992).] Despite the major advances of the past severaldecades in the treatment of serious infections, the incidence andmortality due to sepsis continues to rise. [S. M. Wolff, New Eng. J.Med. 324:486-488 (1991).]

Sepsis is a systemic reaction characterized by arterial hypotension,metabolic acidosis, decreased systemic vascular resistance, tachypneaand organ dysfunction. Sepsis can result from septicemia (i.e.,organisms, their metabolic end-products or toxins in the blood stream),including bacteremia (i.e., bacteria in the blood), as well as toxemia(i.e., toxins in the blood), including endotoxemia (i.e., endotoxin inthe blood). The term “bacteremia” includes occult bacteremia observed inyoung febrile children with no apparent foci of infection. The term“sepsis” also encompasses fungemia (i.e., fungi in the blood), viremia(i.e., viruses or virus particles in the blood), and parasitemia (i.e.,helminthic or protozoan parasites in the blood). Thus, septicemia andseptic shock (acute circulatory failure resulting from septicemia oftenassociated with multiple organ failure and a high mortality rate) may becaused by a number of organisms.

The systemic invasion of microorganisms presents two distinct problems.First, the growth of the microorganisms can directly damage tissues,organs, and vascular function. Second, toxic components of themicroorganisms can lead to rapid systemic inflammatory responses thatcan quickly damage vital organs and lead to circulatory collapse (i.e.,septic shock) and oftentimes, death.

There are three major types of sepsis characterized by the type ofinfecting organism. Gram-negative sepsis is the most common and has acase fatality rate of about 35%. The majority of these infections arecaused by Escherichia coli, Klebsiella pneumoniae and Pseudomonasaeruginosa. Gram-positive pathogens such as the staphylococci andstreptococci are the second major cause of sepsis. The third major groupincludes the fungi, with fungal infections causing a relatively smallpercentage of sepsis cases, but with a high mortality rate.

Many of these infections are acquired in a hospital setting and canresult from certain types of surgery (e.g., abdominal procedures),immune suppression due to cancer or transplantation therapy, immunedeficiency diseases, and exposure through intravenous catheters. Sepsisis also commonly caused by trauma, difficult newborn deliveries, andintestinal torsion (especially in dogs and horses).

Many patients with septicemia or suspected septicemia exhibit a rapiddecline over a 24-48 hour period. Thus, rapid methods of diagnosis andtreatment delivery are essential for effective patient care.Unfortunately, a confirmed diagnosis as to the type of infectiontraditionally requires microbiological analysis involving inoculation ofblood cultures, incubation for 18-24 hours, plating the causativeorganism on solid media, another incubation period, and finalidentification 1-2 days later. Therefore, therapy must be initiatedwithout any knowledge of the type and species of the pathogen, and withno means of knowing the extent of the infection.

It is widely believed that anti-endotoxin antibody treatmentadministered after sepsis is established may yield little benefitbecause these antibodies cannot reverse the inflammatory cascadeinitiated by endotoxin. In addition, the high cost of each antibody(Centoxin HA-1A was expected to cost $3700 per 100 mg dose) would limitphysicians' use of a product where no clear benefit has beendemonstrated. [K. A. Schulman et al., JAMA 266:3466-3471 (1991).] Ofcourse, these endotoxin antibodies only target gram-negative sepsis; noequivalent antibodies exist for the array of gram-positive organisms andfungi.

With new knowledge regarding the effects of endotoxin on hostinflammatory responses, other therapies are being attempted. Forexample, an IL-1 receptor antagonist has been identified that occupiesthe same receptor site as IL-1, but mediates no biological effect.Blockage of the IL-1 receptor with this molecule can reduce mortalityfrom endotoxin shock. [K. Ohlsson et al., Nature 348:550-552 (1990).]While the IL-1 receptor antagonist appears to be well-tolerated, therequired dosage is extremely large (over 100 mg of recombinant proteinper kg of body weight is infused over a period of hours to days). Forhuman therapy, the 8-10 grams of recombinant protein anticipated to berequired is likely to be extremely costly (several thousand dollars).

Clearly, there is a great need for agents capable of preventing andtreating sepsis. It would be desirable if such agents could beadministered in a cost-effective fashion. Furthermore, approaches areneeded to combat all forms of sepsis, not just gram-negative cases.

SUMMARY OF THE INVENTION

The present invention relates to therapeutics for the prevention andtreatment of blood-borne and toxin-mediated diseases, and in particularthe prevention and treatment of sepsis in mammals. The present inventionrelates to compositions and methods for preventing sepsis in high-riskpatients, including immunocompromised patients such as surgical andother hospitalized patients, low birth weight infants, and burn andtrauma victims. The present invention contemplates treatment of mammalshaving symptoms of a systemic septic reaction.

In one embodiment, the present invention contemplates a compositioncomprising a mixture of antibodies directed to TNF and IL-6. In anotherembodiment, the present invention contemplates a method of relievingsymptoms of and rescuing mammals (including humans) from episodes ofacute septicemia and septic shock utilizing a combination preparationcomprising anti-TNF antibodies and anti-interleukin-6 (IL-6) antibodies.The present invention contemplates a method of treatment, comprising:(a) providing: i) a mammal for treatment, ii) a therapeutic preparation,comprising anti-TNF and anti-IL-6 polyclonal antibodies; and (b)administering said preparation to said mammal (e.g., intravenous orparenterally).

Other combinations of antibodies are also contemplated. For example, inone embodiment, the present invention contemplates the use of antibodiesdirected to γIFN in combination with other antibodies, such as anti-TNFantibodies.

Preferably, the polyclonal antibody is reactive with TNF across species.Specifically, the present invention demonstrates that immunization withhuman TNF generates neutralizing antibody capable of reacting withendogenous murine TNF. Thus, the present invention provides anti-TNFantibody that will react with mammalian TNF generally (such as withequine TNF for treatment of equine sepsis).

It is not intended that the present invention be limited to specificpreparations of antibodies. However, polyclonal antibodies arepreferred. Most importantly, it is preferred that the antibodies not becomplement fixing. More specifically, avian antibodies (e.g., chickenantibodies from eggs) are preferred.

The treatment with the combination preparation has the unexpected resultof reducing mortality rates in patients when administered within up tofour hours of the onset of the acute septicemia/septic shock episode.Clearly, the present invention provides an effective approach toprevention and treatment of sepsis.

While acute treatment is contemplated, the present invention alsocontemplates a method of treatment of mammals at risk for developingsepsis, in which a therapeutic preparation comprised of a mixture ofantibodies capable is administered to the at-risk animal prior to orafter the onset of any septic symptoms. In a preferred embodiment, it iscontemplated that the method of the present invention will beadministered intravenously. The present invention contemplates that themethod will be used for such animals as neonatal calves and foals, aswell as human and veterinary surgical patients, trauma, and burnvictims. It is contemplated that the method will be used to treatimmunocompromised patients.

DEFINITIONS

The phrase “symptoms of sepsis” refers to any symptoms characteristic ofa subject with sepsis including but not limited to, arterialhypotension, metabolic acidosis, fever, decreased systemic vascularresistance, tachypnea and organ dysfunction. Sepsis can result fromsepticemia (i.e., organisms, their metabolic end-products or toxins inthe blood stream), including bacteremia (i.e., bacteria in the blood),as well as toxemia (i.e., toxins in the blood), including endotoxemia(i.e., endotoxin in the blood). The term “sepsis” also encompassesfungemia (i.e., fungi in the blood), viremia (i.e., viruses or virusparticles in the blood), and parasitemia (i.e., helminthic or protozoanparasites in the blood). Thus, septicemia and septic shock (acutecirculatory failure resulting from septicemia often associated withmultiple organ failure and a high mortality rate) is a symptom ofsepsis.

Such symptoms are subject to quantitative analysis (e.g., fever, etc.).Some symptoms are readily determined from a blood test (e.g.,bacteremia). The phrase “wherein said symptoms are reduced” refers to aqualitative or quantitative reduction in detectable symptoms, includingbut not limited to a detectable impact on the rate of recovery fromdisease.

The phrase “at risk for sepsis” is herein defined as a subjectpredisposed to the development of sepsis by virtue of the subject'smedical status, including but not limited to such factors as infection,trauma (e.g., abdominal perforation, such as by a gun shot wound),surgery (e.g., intestinal surgery), and invasive procedures (e.g.,placement of a catheter, etc.) and the like.

DESCRIPTION OF THE INVENTION

The present invention relates to therapeutics compositions and methodsfor the prevention treatment of blood-borne and toxin mediated diseases,and in particular the prevention and treatment of sepsis caused byvarious types of organisms in humans as well as other animals. It iscontemplated that the present invention will be used in the treatment ofgram-negative and gram-positive sepsis. Although the invention may beused for treatment of sepsis due to one organism, it may also be used totreat sepsis caused by multiple organisms (e.g., sepsis and/orbacteremia due to gram-negative and gram-positive organisms).

As noted above, the present invention also contemplates treatmentcomprising anti-TNF and anti-IL6 antibody preparations used alone and incombination, as well as other combinations (such as anti-TNF andanti-IFN). The present invention further teaches treatments comprisinganti-TNF and anti-IL6 combination preparations and methods used incombination after the onset of symptoms of blood-borne or toxin-mediateddiseases. In accordance with the present invention, anti-TNF andanti-IL6 combination preparations are administered intravenously,intramuscularly, subcutaneously, intradermally, intraperitoneally,intrapleurally, intrathecally or topically.

Importantly, it is not necessary to the successful use of thecomposition and methods of the present invention that one understand theprecise mechanism by which a therapeutic benefit is achieved. However,it is believed that one of the key mediators of septic shock is tumornecrosis factor (TNF). [R. C. Bone, Ann. Intern. Med. 115:457-469(1991).] Indeed, large doses of TNF [K. J. Tracey et al., Science234:470-474 (1986)] and/or IL-1 [A. Tewari et al., Lancet 336:712-714(1990)] can mimic the symptoms and outcome of sepsis.

Monoclonal antibodies have been found to offer some protection inexperimental animals [S. M. Opal et al., J. Infect. Dis. 161:1148-1152(1990)] but studies in human patients with sepsis have not beenconclusive. The improved results with the combination of antibodiesdescribed herein suggest that neutralization of other mediators—alongwith TNF—is what is needed for a therapeutic benefit.

Experimental

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

EXAMPLE 1 Production of Antibodies to Cytokines TNF Alpha, IL-6, GammaIFN, IL-1B And IL-12 in the Hen

This example involved: (a) preparation of the immunogen andimmunization; (b) purification of anti-cytokine chicken antibodies fromegg yolk (IgY); and (c) detection of anti-cytokine antibodies in thepurified IgY preparations.

(a) Preparation of the immunogen and immunization. The cytokines used toimmunize the hens were purchased from R&D Systems Inc., Minneapolis,Minn. and produced in E. coli. Specifically what was obtained is asfollows: recombinant human Tumor Necrosis Factor Alpha, (TNFα or justTNF), recombinant mouse Interleukin-6, (IL-6), recombinant mouseInterleukin-1 beta, (IL-1B), recombinant mouse Interferon gamma, (gammaIFN), and recombinant mouse Interleukin-12 p40 homodimer (IL-12). Thesecytokines were selected because they are all considered pro-inflammatorycytokines released in response to infection. All the above recombinantproteins were purchased lyophilized without bovine serum albumin (BSA)and designated carrier-free. This was done to prevent an interferingantibody response against BSA after immunization. The lyophilizedcytokine was reconstituted in phosphate-buffered saline pH 7.2-7.5 (PBS)at 50-100 μg/ml and from 2-10 μg of cytokine was used to immunize eachhen. Each hen received one 0.5 ml sub-cutaneous injection containingcytokine with 75 μg Quil A adjuvant (Superfos Biosector, Denmark,distributed by Accurate Chem., Westbury, N.Y.) in PBS. The hens wereimmunized every 2 weeks for at least 3 times then placed on amaintenance immunization schedule where the hens were immunized every4-6 weeks.

(b) Purification of anti-cytokine chicken antibodies from egg yolk(IgY). Groups of eggs were collected per immunization group at least 3-5days after the last booster immunization. The chicken yolkimmunoglobulin (IgY) was extracted by a two-step polyethylene glycol(PEG) 8000 method performed according to a modification of the procedureof Polson et al., Immunol. Comm., 9:495 (1980). The yolks were separatedfrom the whites and the yolks were placed in a graduated cylinder. Thepooled yolks were blended with 4 volumes of PBS and PEG was added to aconcentration of 3.5%. When the PEG was dissolved, the protein and lipidprecipitates that formed were pelleted by centrifugation at 9,000×g for15 minutes. The supernatants were decanted and filtered through 4 layersof gauze to remove the floating particulates and a second PEG step wasperformed by adding PEG to a final concentration of 12% (thesupernatants were assumed to contain 3.5% PEG). After a secondcentrifugation, the supernatants were discarded and the IgY pellets wereresuspended in PBS at approximately ⅙ the original yolk volume. IgYsextracted from eggs from unimmunized hens (designated preimmune IgY)served as control IgY. The concentration of the fractionated IgY's wereestimated by measuring the absorbance at 280 nm (an optical density at280 nm of 1.3 equals 1 mg of IgY/ml. The antibody concentrations wereabout 25-30 mg/ml.

(c) Detection of anti-cytokine antibodies in the purified IgYpreparations. In order to determine if anti-cytokine response wasgenerated and to determine relative levels of the response,enzyme-linked immunosorbent assays (ELISA) were performed. Briefly,ninety-six well Falcon Pro-bind micro-titer plates were coated overnightat 4° C. with 100 μl/well of cytokine at 0.1-1.0 μg/ml PBS. The wellsare then blocked with PBS containing 1-3% BSA and 0.05% Tween 20 andincubated for about 1 hour at 37 deg C. The blocking solution wasremoved and the immune or preimmune IgY was diluted in PBS containingBSA and the plates were incubated for 1 hour at 37 deg C. The plateswere washed 3 times with PBS containing 0.05% Tween 20 and three timeswith PBS alone. Alkaline phosphatase-conjugated anti-chicken IgG wasdiluted 1:1000 in PBS containing 1% BSA and 0.05% Tween 20, added to theplates and incubated 1 hour at 37 deg C. The plates were washed as aboveand p-nitrophenyl phosphate at 1 mg/ml in 0.05 M Na₂CO₃, pH 9.5, 10 mMMgCl₂ was added. The plates were read in a Dynatech plate reader at 410nm about 30 minutes after substrate addition. Good antibody titers(reciprocal of the highest immune IgY generating a signal about 3-foldhigher than that of preimmune) ranging from 10,000 to 50,000 wasgenerated in all the cytokines except IL-1B which did not elicit anantibody response using the amounts of antigen used to immunize thehens. The level of antibody response in the hens against most of thecytokines considering the low amounts of antigen used for immunizationindicate that mammalian cytokines are very immunogenic in the hens andis a well-suited system to generate mammalian cytokine antibodies.

EXAMPLE 2 Neutralization of the In Vivo Effects of Endotoxin/D-GalN byAvian Anti-TNFα Antibody

Endotoxin (LPS) can trigger a lethal reaction in vivo. In order todetermine whether avian anti-TNF antibody is capable of neutralizing thelethal effects of endotoxin, a well-characterized and accepted murinemodel of endotoxic shock was utilized. [C. Galanos et al., Proc. Natl.Acad. Sci. USA 76:5939-5943 (1979).] The example involved: (a) use of alethal dose of endotoxin in galactosamine (D-GalN)-sensitized mice; (b)neutralization of endotoxin lethality by premixture with avian anti-TNFantibody; and (c) rescue of lethality by administration of aviananti-TNF antibody at time points subsequent to LPS administration.

(a) A lethal dose (10-100 ng) of endotoxin in galactosamine-sensitizedmice was administered to C3H/HEN (Charles River, Wilmington, Mass.) micethat were co administered 18 mg of D-galactosamine-HCl in 200 μl ofphosphate buffered saline (PBS). The latter compound is a specifichepatotoxic agent that increases the sensitivity of experimental animalsto endotoxin several thousand-fold. [C. Galanos et al., Proc. Natl.Acad. Sci. USA 76:5939-5943 (1979).] To accomplish this determination,E. coli 0111:B4 LPS (List Biological Laboratories, Campbell, Calif.) inPBS was injected intraperitoneally, along with 18 mg of D-galactosamine(Sigma Corp.).

(b) Neutralization of endotoxin lethality by premixture with chickenanti-TNF antibody was performed mixing 10-100 ng of E. coli 0111:B4 LPSwith 4-8 mg of polyclonal anti-TNF antibody (prepared as described inExample 1). The results are shown in Table 1. Note that the use ofanti-TNF antibody premixed with endotoxin resulted in a significantlyreduction in the lethality as compared to PBS and Preimmune premixcontrols, with a 76% survival rate for the anti-TNF antibody premixcontrasted with 0% survival rates for both the PBS and Preimmune premixcontrols.

(c) Rescue of lethality by administration of chicken anti-TNF antibodywas attempted at time points subsequent to LPS administration. Survivalwas assessed at 5, 10, 30 and 60 minutes post induced shock. The resultsare shown in Table 1, which describes the use of avian anti-TNF of thepresent invention, as administered at various temporal intervals to micethat had been induced with septic shock syndrome. Note that theshock-induced mice which were treated with the avian anti-TNF antibodyexperienced a significant survival rate of 61% for a period up to 30minutes post-shock. The anti-TNF survival rate of 20% at 60 minutes postshock further displays a low rate of lethality as compared with the 0%survival rates for both the PBS and Preimmune premix controls.

The results of this experiment demonstrates that avian anti-TNF antibodyneutralizes the lethal effect of endotoxin in vivo and suggest thatavian anti-TNF antibody will be useful in preventing sepsis as well asin treating the early stages of sepsis due to gram-negative bacteria.

TABLE 1 No. Of Survivors/ Treatment No. Of Expt. No. Tested % SurvivalPBS (premix) 2 0/7  0 Preimmune (premix) 4 0/20 0 Anti-TNF (premix) 313/17  76 Anti-TNF (5 min. post) 2 5/10 50 Anti-TNF (10 min. post) 13/5  60 Anti-TNF (30 min. post) 3 8/13 61 Anti-TNF (60 min. post) 2 2/1020

EXAMPLE 3 Neutralization of the In Vivo Effects of Endotoxin/D-GalN byAvian Anti-TNF Antibody and Anti-IL-6 Antibody Administered inCombination

As mentioned earlier, LPS can trigger a lethal reaction in vivo. Inorder to determine whether an avian anti-TNF antibody in combinationwith another anti-cytokine antibody can increase the survival ratepost-challenge beyond that of anti-TNF antibody alone, severalcombination therapies were tested. The combination therapies were testedin a well-characterized and accepted murine model of endotoxic shockusing LPS and galactosamine. [C. Galanos et al., Proc. Natl. Acad. Sci.USA 76:5939-5943 (1979).] The example involved: (a) use of a lethal doseof endotoxin in galactosamine-sensitized mice; and (b) rescue oflethality by administration of anti-TNF antibody in combination withanti-IL-6 antibody at time points subsequent to LPS administration.

(a) A lethal dose of endotoxin in galactosamine-sensitized mice wasadministered according to the method referred to in Example 2 part (a)above. Again, 10-100 ng of LPS with 18 mg of DGalN was an effectivelethal dose in 20-22 g C3H/HEN mice.

(b) Rescue of lethality by administration of anti-TNF antibody incombination with anti-IL-6 antibody was attempted at various time pointssubsequent to LPS/D-GalN administration. In these experiments 4-8 mgs ofantibody in PBS were administered i.p. When using the anti-TNFantibody/anti-IL6 antibody combination therapy, the antibodies weremixed at a 1:1 ratio and half the amount of each individual antibody wasused (i.e., as compared to the treatments where each antibody is usedalone). Survival was assessed at 60, 120, 180 and 240 minutes after theinduced shock.

The results are shown in Table 2, which shows the results following theuse of the avian anti-TNF antibody and anti-IL-6 antibody combination ofthe present invention, as administered to shock-induced mice at varioustemporal intervals. Of particularly note are the percentage of survivors(82%) for the avian anti-TNF and anti-IL6 combination administered at 60minutes post shock, as contrasted with the significantly lower survivornumbers (25%) for the Preimmune premix controls at 60 minutes postshock. Significantly, anti-TNF, or anti-IL-6 administered alone at 60minutes post shock showed only a 40% survival rate. Of further note isthe fact that the avian anti-TNF and anti-IL6 combination survivorsadministered with the anti-TNF and anti-IL6 combination (indicated as“Combo”) at 120 minutes after the onset of the of the septic reactionstill showed significant survival rates of 48%, when compared with thepreimmune control of 18% and 25% at 60 and 120 minutes post shock,respectively.

TABLE 2 No. Of Survivors/ % Treatment No. Of Expt. No. Tested SurvivalPBS 4  5/23 22 Preimmune (60 min. post) 2  2/11 18 Preimmune (120 min.post) 1 2/8 25 Anti-TNF (60 min. post) 1 2/5 40 Anti-IL-6 (60 min. post)1 2/5 40 Combo (60 min. post) 4 18/22 82 Combo (120 min. post) 5 15/3148 Combo (180 min. post) 1 2/6 33 Combo (240 min. post) 1 2/8 25The results indicate that the protection afforded by the combinationtherapy begins to wane at 180 to 240 minutes post-challenge. Clearly,the results of this experiment demonstrate that avian anti-TNF antibodyadministered in combination with anti-IL-6 antibody can rescue mammalsfrom the lethal effect of endotoxin in vivo, and is more effective intreating sepsis post-challenge than anti-TNF antibody alone. Theseresults also suggest that an avian anti-TNF antibody and anti-IL-6combination therapy will be useful in preventing or treating sepsis inthe later stages of development in mammals, including but not limited tohumans.

EXAMPLE 4 Failure to Neutralize the In Vivo Effects of Endotoxin byUsing Combinations of Anti-Gamma IFN and Anti-IL-6, or Anti-Gamma IFNand Anti-TNF

In the above examples, a composition comprising a combination ofpolyclonal antibodies directed to TNF and IL-6 was shown to be capableof blocking the downstream cascade of sepsis in an animal model. In thisexample, other anti-cytokine IgYs were tested: (a) a polyclonal antibodyagainst gamma IFN; (b) a combination of polyclonal antibodies directedto gamma IFN and IL-6; and (c) a combination of polyclonal antibodiesdirected to gamma IFN and TNF. Again, the LPS/D-GalN animal model wasemployed as described in Examples 2 and 3. The model was tested in themanner above (i.e., in an attempt to rescue animals post-induced shock).The results are shown in Table 3.

TABLE 3 No. Of Survivors/ % Treatment No. Of Expt. No. Tested SurvivalPreimmune (60 min. post) 2  2/11 18 Anti-Gamma IFN 1 0/3 0 (60 min.post) Anti-Gamma IFN + Anti- 1 1/3 33 IL-6 (60 min. post) Anti-TNF +Anti-Gamma 1 0/3 0 IFN (60 min. post)

The results from Table 3 show that anti-gamma IFN antibody alone as wellas combinations of this antibody with 1) antibodies to IL-6, and 2)antibodies to TNF, are unable to provide significant protection in themouse 60 minutes post-challenge. In contrast to the anti-TNF/anti-IL-6combination therapy, the anti-IFN combination therapies (tested in thisexample) do not appear to be capable of blocking the downstream cascade.

EXAMPLE 5 Prevention of the In Vivo Effects of Endotoxin by Pretreatment

The anti-cytokine antibodies either singly or in combination were testedin another type of endotoxin shock model. This model uses only very highlevels of LPS without D-Gal-N. This model is thought to be distinct fromthe LPS/D-Gal-N shock model in that cytokine antibodies effective inthis model are ineffective in the other models. This model is alsoconsidered very aggressive, and the onset of septic shock is quiterapid. The example involved: (a) use of lethal doses of LPS in the mice;(b) neutralization of endotoxin lethality by a pretreatment orpremixture with avian anti-TNF alpha antibody, anti-Gamma INF, andanti-IL-6; and (c) rescue of lethality by administration of combinationtherapy of avian anti-TNF alpha antibody, anti-Gamma INF, and anti-IL-6post LPS challenge.

(a) A lethal dose of 1.2 mg of LPS (Salmonella enteritidis) (SigmaChemical Co., St. Louis, Mo.) was administered intraperitoneally to18-20 g C57BL/6 mice (Charles River) in 400 ml of PBS. This LPS onlyendotoxin shock model is described in J. Rothe et al. [Nature364:798-802 (1993)].

(b) Neutralization of endotoxin lethality by a pretreatment withcytokine antibody was performed by interperitonally injecting 400 ml PBScontaining 1.2 mg of LPS 1 hour later. In the premix studies the sameamounts of cytokine antibody and LPS were premixed then immediatelyadministered intraperitoneally. The results are shown in Table 4.Preimmune IgY was found to be ineffective in protecting the mice. Inaddition, pretreatment with anti-IL-12 was also ineffective. Incontrast, anti-TNF and anti-gamma IFN as a pretreatment were veryeffective in protecting the animals with survival rates of 88% and 100%,respectively. Moreover, anti-TNF, anti-IL-6 and anti-gamma IFN as apremix were also effective, with survival rate of 77% and 88%.

TABLE 4 No. Of Survivors/ % Treatment No. Of Expt. No. Tested SurvivalPreimmune 3  1/11 9 (60 min. pretreatment) Anti-TNF 2 7/8 88 (60 min.pretreatment) Anti-IL-12 1 1/3 33 (60 min. pretreatment) Anti-Gamma IFN1 3/3 100 (60 min. pretreatment) Preimmune (premix) 2 0/6 0 Anti-TNF(premix) 2 3/6 50 Anti-IL-6 (premix) 3 7/9 77 Anti-Gamma IFN (premix) 38/9 88

(c) The rescue of lethality post LPS challenge using cytokine antibodieseither singly or in combination is shown in Table 5. In all anticytokine therapies shown in Table 5, the final amount of IgYadministered to the mice was the same (4-8 mg). The combinationtherapies were comprised of ½ or ⅓ of each individual anti-cytokine inthe dual therapies or the triple therapies respectively. PBS at 5 minutepost challenge or preimmune at 5 and 10 minute post challenge were, asexpected, unable to protect the mice. In addition, either anti-TNF aloneat 5 and 15 minute post challenge or combination therapies ofanti-IL-6/Gamma IFN (5 minute post) or anti-TNF/anti-IL-6 (5 and 15minute post) could not significantly protect the animals. Interestingly,a triple combination of anti-TNF/anti-IL-6/anti-Gamma INF was completelyeffective at 5 minute post challenge but not at 15 minute postchallenge.

These results indicate in this model anti-Gamma IFN may be beneficial inaddition to anti-TNFα and anti-IL-6 combination found to be effective inthe LPS/D-GalN shock model.

TABLE 5 No. Of Survivors/ % Treatment No. Of Expt. No. Tested SurvivalPBS (5 min. post) 1 0/4 0 Preimmune 3  0/12 0 (5 and 15 min. post)Anti-TNF 2 0/9 0 (5 and 15 min. post) Anti-IL-6/Gamma IFN 1 1/3 33 (5min. post) Anti-TNF/Anti-IL-6/Anti- 2 6/6 100 Gamma IFN (5 min. post)Anti-TNF/Anti-IL-6/Anti- 1 1/3 33 Gamma IFN (15 min. post)Anti-TNF/Anti-IL-6 2 0/9 0 * Survival at least 24 hours post-challenge(5 and 15 min. post).

EXAMPLE 6 Neutralization of the In Vivo Effects of Endotoxin in aGram-Positive Model

In the above examples, the combination of polyclonal antibodies directedto TNF and IL-6 was shown to be capable of blocking the downstreamcascade of sepsis in an animal model involving gram negative sepsis. Inthis example, polyclonal antibodies directed to TNF were tested aloneusing a gram positive sepsis model. The gram-positive sepsis model wasperformed as described by S. Q. DeJoy et al., J. Infect. Dis.169:150-156 (1994). To carry out tests on the model, 20-22 g C3H/HeN(Charles River) mice were treated i.p. with heat-killed Staph. aureusbacteria in the presence of galactosamine. The mice were given 0.1 ml ofthe killed bacteria at the optical density at A600 of 90-100 mixed with0.1 ml PBS with 18 mg of galactosamine. The mice are given i.p. 400 ulof PBS containing 4-8 mgs of IgY. IgY. The results (using the premixtreatment format described in Example 2) are shown in Table 6.

TABLE 6 No. Of Survivors/ Treatment No. Of Expt. No. Tested % SurvivalUntreated 1 0/3 0 Preimmune (premix) 1 0/3 0 Anti-TNF (premix) 1 3/4 75The results show that using anti-TNF antibody provided significantprotection. The results of the post-challenge treatment are shown inTable 7. Both preimmune and anti-IL-6 antibody administered alone 5minutes post-challenge were unable to protect the animals (i.e.,survival rates were 20-25%). On the other hand anti-TNF antibody showedbetter protection, with the combination (“Combo”) therapy (using equalamounts of anti-TNF antibody and anti-IL-6 antibody) gave the bestsurvival rates at 89% (at 48 hours post-challenge). These resultsindicate that an anti-cytokine combination therapy (in accordance withthe teachings of the present invention) against gram-positive sepsis isan effective treatment.

TABLE 7 No. Of Survivors/ Treatment No. Of Expt. No. Tested % SurvivalPreimmune (5 min. post) 2 1/5 20 Anti-IL-6 (5 min. post) 2 2/8 25Anti-TNF (5 min. post) 2 4/8 50 Combo (5 min. post) 2 8/9 89

1. A method of treatment, comprising: a) providing: i) a mammal, ii) atherapeutic preparation, comprising anti-TNF and anti-IL-6 antibodies;and b) administering said preparation to said mammal.
 2. The method ofclaim 1, wherein said mammal is a human.
 3. The method of claim 1,wherein said administering is performed intravenously.
 4. The method ofclaim 1, wherein said administering is performed orally.
 5. The methodof claim 1, wherein said administering is performed parenterally.
 6. Themethod of claim 2, wherein said human has symptoms of sepsis.
 7. Themethod of claim 2, wherein said human is at risk for sepsis.
 8. Themethod of claim 1, wherein said antibodies are polyclonal antibodies. 9.The method of claim 8, wherein said polyclonal antibodies are avianantibodies.
 10. The method of claim 9, wherein said avian antibodies arechicken antibodies.
 11. The method of claim 10, wherein said chickenantibodies are derived from chicken eggs.
 12. A method of treatment,comprising: a) providing: i) a mammal with symptoms of gram-positivesepsis, ii) a therapeutic preparation, comprising polyclonal anti-TNFand anti-IL-6 antibodies; and c) administering said preparation to saidmammal under conditions wherein said symptoms are reduced.
 13. Themethod of claim 12, wherein said mammal is a human.
 14. The method ofclaim 12, wherein said administering is performed intravenously.
 15. Themethod of claim 12, wherein said administering is performed orally. 16.The method of claim 12, wherein said administering is performedparenterally.
 17. The method of claim 12, wherein said polyclonalantibodies are avian antibodies.
 18. A method of treatment, comprising:a) providing: i) a human with symptoms of gram-positive sepsis, ii) atherapeutic preparation, comprising polyclonal anti-TNF and anti-IL-6antibodies; and d) administering said preparation to said human underconditions wherein said symptoms are reduced.
 19. The method of claim18, wherein said polyclonal antibodies are avian antibodies.
 20. Atherapeutic preparation suitable for administration to a human,comprising polyclonal anti-TNF and anti-IL-6 antibodies.
 21. Thepreparation of claim 20, wherein said antibodies are in equal amounts.22. The preparation of claim 20, wherein said polyclonal antibodies areavian antibodies.